Summary: Tuberculosis is among the most important global public health concerns with more than 2 million people dying from this disease each year. The current tuberculosis vaccine, BCG, has questionable efficacy and is counterindicated in some of the target populations. Recent studies have indicated that DNA vaccines may be promising as new mycobacterial vaccines candidates. We have created 35 DNA vaccines expressing different mycobacterial antigens. These tuberculosis DNA vaccines have been generated to express TPA-fused antigens, ubiquitin-conjugated proteins and native antigens. All of these vaccines have been tested by intramuscular inoculation,either singly or in combination, for their capacity to induce protective, cytokine, and humoral immune responses in mice. Based on the results of these studies, we have concluded the following: 1. At least 12 tuberculosis DNA vaccines that we have tested induce protective immune responses in murine primary aerogenic infection models. However, the level of protection for single vaccines is less than the level of protection evoked by BCG. 2. Tuberculosis DNA vaccines encoding mycobacterial antigens fused to a TPA signal signal are more immunogenic than DNA vaccines encoding the native protein. TB DNA vaccines encoding mycobacterial antigens fused to ubiquitin induce primarily high levels of cell-mediated immunity and low humoral responses. 3. Combinations of TB DNA vaccines do evoke a larger protective response than single vaccines. The level of protective immunity elicited by a 10 component TPA TB DNA vaccines combination and a 10 component ubiquitin DNA vaccine combination was equivalent to the BCG response in long-term studies. In three studies, mice immunized with either the TPA TB DNA vaccine combination, the ubiquitin vaccine cocktail, or BCG survived as much as 8-fold longer than naive mice after an aerogenic challenge with virulent M. tuberculosis. 4. Thus far, we have been unable to boost the BCG response by priming with or concurrent administration of the DNA vaccine combinations. 5. The effectiveness of TB DNA vaccination has also been evaluated in a mouse model of latent tuberculosis. Immunization with the DNA vaccine combinations does not prevent reactivational disease in this model but does have a modest effect on reducing the growth of a secondary challenge (exogenous reinfection). We recently submitted a proposal to the NIH TB vaccine testing program to test our DNA vaccine combination in a guinea pig TB model. Because of our success in the mouse model, the proposal was approved and the effectiveness of our DNA vaccine preparation is currently being assessed in guinea pigs. To further define the specific immune responses responsible for protective immunity to tuberculosis, we have evaluated the susceptibility to TB infection of mice deficient in the CIITA gene. The CIITA gene is the IFN-regulated master transactivator of MHC class II expression. In low dose aerogenic challenge studies, we have shown that CIITA knockout mice are highly susceptible to tuberculosis. The median time to death for these KO mice is about 60 days while wild-type mice survive about 300 days after a low dose challenge. FACS analyses of splenocytes from KO mice indicate that only CD8 T cells are induced after TB infection of these mice and not the CD4 T cells that are largely responsible for controlling TB infections. As a result of their failure to generate immune CD4 cells, IFN-gamma levels in the KO mice (in response to the infection) are dramatically reduced relative to wild-type mice. This study demonstrates that the MHC class II pathway is an essential component of the protective immune response to TB and that new vaccines must be designed to stimulate class II immunity.